Gram-negative bacteria assemble a variety of surface structures, including the hair-like organelles known as pili or fimbriae. Pili typically function in adhesion and mediate interactions with various surfaces, with other bacteria, and with other types of cells such as host cells. The chaperone/usher (CU) pathway assembles a widespread class of adhesive and virulence-associated pili. Pilus biogenesis by the CU pathway requires a dedicated periplasmic chaperone and integral outer membrane protein termed the usher, which forms a multifunctional assembly and secretion platform. This review addresses the molecular and biochemical aspects of the CU pathway in detail, focusing on the type 1 and P pili expressed by uropathogenic as model systems. We provide an overview of representative CU pili expressed by and , and conclude with a discussion of potential approaches to develop antivirulence therapeutics that interfere with pilus assembly or function.

Catheter-associated urinary tract infection (CAUTI) is the most common healthcare-associated infection and cause of secondary bloodstream infections. Despite many advances in diagnosis, prevention and treatment, CAUTI remains a severe healthcare burden, and antibiotic resistance rates are alarmingly high. In this review, current CAUTI management paradigms and challenges are discussed, followed by future prospects as they relate to the diagnosis, prevention, and treatment. Clinical and translational evidence will be evaluated, as will key basic science studies that underlie preventive and therapeutic approaches. Novel diagnostic strategies and treatment decision aids under development will decrease the time to diagnosis and improve antibiotic accuracy and stewardship. These include several classes of biomarkers often coupled with artificial intelligence algorithms, cell-free DNA, and others. New preventive strategies including catheter coatings and materials, vaccination, and bacterial interference are being developed and investigated. The antibiotic pipeline remains insufficient, and new strategies for the identification of new classes of antibiotics, and rational design of small molecule inhibitor alternatives, are under development for CAUTI treatment.

The chaperone-usher (CU) pathway assembles organelles termed pili or fimbriae in Gram-negative bacteria. Type 1 pili expressed by uropathogenic Escherichia coli are prototypical structures assembled by the CU pathway. Biogenesis of pili by the CU pathway requires a periplasmic chaperone and an outer membrane protein termed the usher (FimD). We show that the FimD C-terminal domains provide the high-affinity substrate binding site, but that these domains are masked in the resting usher. Domain masking requires the FimD plug domain, which serves as a switch controlling usher activation. We demonstrate that usher molecules can act in trans for pilus biogenesis, providing conclusive evidence for a functional usher oligomer. These results reveal mechanisms by which molecular machines such as the usher regulate and harness protein-protein interactions, and suggest that ushers may interact in a cooperative manner during pilus assembly in bacteria.

Pathogenic bacteria such as Escherichia coli assemble surface structures termed pili, or fimbriae, to mediate binding to host‐cell receptors. Type 1 pili are assembled via the conserved chaperone usher pathway. The outer‐membrane usher FimD recruits pilus subunits bound by the chaperone FimC via the periplasmic N‐terminal domain of the usher. Subunit translocation through the β‐barrel channel of the usher occurs at the two C‐terminal domains (which we label CTD1 and CTD2) of this protein. How the chaperone–subunit complex bound to the N‐terminal domain is handed over to the C‐terminal domains, as well as the timing of subunit polymerization into the growing pilus, have previously been unclear. Here we use cryo‐electron microscopy to capture a pilus assembly intermediate (FimD–FimC–FimF–FimG–FimH) in a conformation in which FimD is in the process of handing over the chaperone‐bound end of the growing pilus to the C‐terminal domains. In this structure, FimF has already polymerized with FimG, and the N‐terminal domain of FimD swings over to bind CTD2; the N‐terminal domain maintains contact with FimC–FimF, while at the same time permitting access to the C‐terminal domains. FimD has an intrinsically disordered N‐terminal tail that precedes the N‐terminal domain. This N‐terminal tail folds into a helical motif upon recruiting the FimC‐subunit complex, but reorganizes into a loop to bind CTD2 during handover. Because both the N‐terminal and C‐terminal domains of FimD are bound to the end of the growing pilus, the structure further suggests a mechanism for stabilizing the assembly intermediate to prevent the pilus fibre diffusing away during the incorporation of thousands of subunits. Support or Funding Information R01GM062987 Van Andel Institute Graduate School This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Purpose: To determine the composition and site of initiation of bacterial biofilm location on indwelling urinary catheters, and to track biofilm progression over time.Materials & Methods: Indwelling urinary catheters were collected from two tertiary care centers following removal from patients. Indwelling time was noted, and catheters were deidentified. Catheters were sectioned, stained for biofilms, and analyzed using spectrophotometry and visualization. Biofilm colonization patterns were analyzed using descriptive statistical analyses and bacterial composition was determined using next-generation sequencing.Results: 33 catheters from 26 males and 7 females were collected with indwelling times ranging from 15 minutes to 43 days and analyzed. Biofilm colonization was consistently high on the region of the balloon throughout indwelling times. After week 1, the distal third of the catheter had higher biofilm colonization than the proximal third (week 2: p=0.034). At all indwelling times, the intraluminal surface of the catheter had greater biofilm colonization than the outer surface. Nextgeneration sequencing detected potential uropathogenic bacteria in 10 of 10 analyzed samples. Conclusions:The catheter balloon, its distal aspect, and its lumen were the predominant locations of biofilms, comprised of uropathogenic bacteria. Strategies to prevent or treat biofilms should be targeted to these areas.

The PapC usher, a β-barrel pore in the outer membrane of uropathogenic Escherichia coli, is used for assembly of the P pilus, a key virulence factor in bacterial colonization of human kidney cells. Each PapC protein is composed of a 24-stranded β-barrel channel, flanked by N- and C-terminal globular domains protruding into the periplasm, and occluded by a plug domain (PD). The PD is displaced from the channel towards the periplasm during pilus biogenesis, but the molecular mechanism for PD displacement remains unclear. Two structural features within the ß-barrel, an α-helix and β5-6 hairpin loop, may play roles in controlling plug stabilization. Here we have tested clusters of residues at the interface of the plug, barrel, α-helix and hairpin, which participate in electrostatic networks. To assess the roles of these residues in plug stabilization, we used patch-clamp electrophysiology to compare the activity of wildtype and mutant PapC channels containing alanine substitutions at these sites. Mutations interrupting each of two salt bridge networks were relatively ineffective in disrupting plug stabilization. However, mutation of two pairs of arginines located at the inner and the outer surfaces of the PD resulted in an enhanced propensity for plug displacement. One arginine pair involved in a repulsive interaction between the linkers that tether the plug to the β-barrel was particularly sensitive to mutation. These results suggest that plug displacement, which is necessary for pilus assembly and translocation, may require a weakening of key electrostatic interactions between the plug linkers, and the plug and the α-helix.

A significant drop in post-PFC PSA may indicate elimination of the index lesion. Patients who are likely to recur biochemically have a significantly higher PSAV compared to those who do not recur. Nadir PSA of less than 2 ng/ml may be considered the new normal PSA in focal cryotherapy (hemiablation) follow-up.

Focal cryotherapy and moderate-cold (≥-76°C) temperature were associated with favorable sexual function relative to total cryotherapy and very cold temperature, respectively. No significant differences in urinary function or bowel habits were observed between groups.